Bovine Herpesvirus 1 Infection Research Articles

Detection of Infectious Bovine Rhinotracheitis in Natural Cases of Bovine Abortion by PCR and Histopathology Assays

Bovine herpesvirus 1 (BHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), is considered to be the most common viral pathogen found in bovine. Bovine Herpesvirus 1(BHV-1) belongs to the genus of Varicellovirus and the family of Herpesviridae which contains three main gB, gC and gD genes. The objective of this study was to determine the relation between type of macroscopic lesions and bovine herpesvirus-1(BHV-1) infection; hence the aborted fetus specimens were initially prepared for PCR test. In this study, 23 of fetus samples, which were transferred to Veterinary Laboratories during 2009-2012, were assessed for BHV-1 with PCR in lung and spleen tissues and all of positive samples undergone histopathological study. The results indicated myocarditis and necrosis of cardiac tissue in 8 cases (42.1%), and 6 cases encompassed interstitial lymphocytic pneumonia (28.57%), whereas 5 cases revealed bronchopneumonia (23.8%) and in 8 cases (38.09%) severe interstitial edema was observed. In addition, the lesions may be associated with BHV-1, so that in brain tissue 9 cases (36 %) possessed encephalitis and necrosis, 8 cases (38.09%) indicated interstitial nephritis, 2 cases (9.52%) developed acute tubular necrosis, 2 cases (9.52%) revealed vacuolar degeneration inrenal tubular epithelium, 1 case had (4.76%) renal hemorrhage and in 3 cases (14.28%) renal autolysis was observable. In liver, 10 cases (47.61%) were affected to lymphocytic hepatitis, 5 cases (23.8%) possessed necrotic hepatitis, 6 cases (28.57%) of multifocal coagulative necrosis without inflammatory reaction and 3 cases (14.28%) with lymphocytic portal hepatitis were observed. In this assessment, 10 specimens (52.17%) of spleen tissues revealed necrosis and lymphatic tissues emptying and in 10 cases (100%) severe enteritis, which may be associated with IBR, were recorded. This study suggests based on lesions in several organs induced by IBR virus and real time PCR confirmation there is direct correlation between virus and lesions, and in order to obtain more reliable results of IBR infection the histopathological examination may be efficient.

Development of latex agglutination test for rapid detection of antibodies against Bovine herpesvirus 1 and Bovine herpesvirus 5 in cattle

A latex agglutination test (LAT) based on sensitized polystyrene beads with inactivated Bovine herpesvirus 1 (BHV-1) particles was developed to detect serum antibodies against BHV-1 and Bovine herpesvirus 5 (BHV-5). Compared with a virus neutralization test using 252 serum samples, the sensitivity and specificity were 94.7% and 97.5%, respectively. Finally, 512 clinical serum samples from 3 major cattle-breeding provinces were monitored, and the overall positive rate was 34.57% (95% confidence interval: 30.45-38.69%). The results suggest that LAT has the potential to be a rapid method for the diagnosis of BHV-1 and BHV-5 infection in cattle herds.

Protective effects against abortion and fetal infection following exposure to bovine viral diarrhea virus and bovine herpesvirus 1 during pregnancy in beef heifers that received two doses of a multivalent modified-live virus vaccine prior to breeding

To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection. Randomized controlled clinical trial. 33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves. 20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days. After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers. Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.

Bovine herpesvirus-1 VP8 interacts with DNA damage binding protein-1 (DDB1) and is monoubiquitinated during infection

VP8 is the most abundant tegument protein of bovine herpesvirus-1 (BHV-1). In the present study DNA damage binding protein 1 (DDB1) was identified as interacting partner of VP8. MALDI-TOF mass spectroscopy analysis of proteins co-immunoprecipitated with VP8 identified DDB1 as a protein interacting with VP8. The interaction between VP8 and DDB1 was confirmed based on co-immunoprecipitation and co-localization in both VP8-transfected and BHV-1 infected cells. DDB1 was distributed both in the nucleus and the cytoplasm with some nuclear speckles prior to BHV-1 infection, became perinuclear by 4h and was predominantly nuclear at 5h post infection, where it co-localized with VP8. In contrast, in cells infected with a UL47 deletion mutant DDB1 remained cytoplasmic throughout the course of infection. This suggests that VP8 mediates nuclear re-localization of DDB1. Finally, VP8 was shown to be monoubiquitinated both in VP8-transfected and BHV-1-infected cells. These data suggest that BHV-1 VP8 interacts with DDB1-CUL4 E3 ubiquitin ligase, which correlates to monoubiquitination of this viral protein.

Fatores de risco associados à infecção pelo Herpesvírus Bovino Tipo 1 em rebanhos bovinos leiteiros da região Amazônica maranhense

O presente estudo teve como objetivo determinar a frequência e os fatores de risco para a infecção pelo Herpesvírus Bovino Tipo 1 (BoHV-1) em fêmeas bovinas leiteiras na região Amazônica maranhense. Amostras de soro de animais não vacinados contra o BoHV-1 e provenientes de 40 propriedades foram submetidas à técnica de ELISA indireto. Em cada propriedade avaliada, aplicou-se ainda um questionário epidemiológico para investigar os fatores de risco que poderiam estar associados à infecção pelo vírus. Das 400 amostras de soro analisadas, 69,25% (n = 277) foram reagentes com a detecção de bovinos sorologicamente positivos em 100% (n = 40) das propriedades. Dentre os fatores de risco avaliados, faixa etária entre 3 a 7 anos, ausência de assistência veterinária e sinais respiratórios apresentaram significância estatística (P < 0,05) associada à infecção pelo BoHV-1. Os resultados do estudo indicam que a frequência de anticorpos contra o BoHV-1 foi elevada na região Amazônica maranhense e que os fatores relacionados às características das propriedades e ao manejo foram associados ao risco de infecção pelo BoHV-1.

Seroprevalence and Risk Factors of Bovine Herpesvirus 1 Infection in Cattle Herds in the State of Paraná, Brazil

Epidemiological data describing bovine herpesvirus 1 (BoHV-1) infection in Brazilian cattle herds are scarce. A cross-sectional study was conducted in the state of Paraná between December 2001 and July 2002 with the objective of estimating the apparent prevalence of BoHV-1-seropositive herds and animals and identifying the potential risk factors for infection in farms with breeding animals in the state of Paraná in Southern Brazil. The state was divided into seven regions based on the livestock production dynamics of the different areas. Sampling was performed in two stages. Initially, herds were randomly selected, followed by a random selection of animals within the selected herds. Blood samples were collected from 14803 females, aged ≥24 months, from 2018 BoHV-1 non-vaccinated herds. Serum samples were tested for antibodies against BoHV-1 using an indirect enzyme-linked immunosorbent assay (ELISA). The apparent prevalence of seropositivity in the herds and animals in Paraná was 71.3% (95% CI: 69.3-73.3) and 59.0% (95% CI: 56.2-61.8), respectively. Multiple logistic regression analysis revealed that risk factors associated with the presence of the infection were as follows: beef herds [OR = 1.58 (1.12-2.23)], natural service [OR = 1.48 (1.02-2.14)], purchase of animals [OR = 1.90 (1.52-2.37)], pasture rental [OR = 2.24 (1.51-3.33)], existence of calving pens [OR = 1.56 (1.20-2.03)] and records of abortion in the last 12 months [OR = 1.45 (1.08-1.95)]. These results indicate that BoHV-1 infection is widespread in the state of Paraná.

Bovine Herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

Recombinant MVA Expressing Secreted Glycoprotein D of BoHV-1 Induces Systemic and Mucosal Immunity in Animal Models

Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.

Dynamics of individual animal Bovine Herpes Virus-1 antibody status on 9 commercial dairy herds

Bovine Herpes Virus 1 (BoHV-1) is an important viral disease of cattle worldwide. In endemically infected herds, there is an incomplete understanding of the epidemiology of BoHV-1 infection. We describe the dynamics of animal-level BoHV-1 antibody status on 9 endemically infected commercial dairy herds, based on the results of serial milk antibody testing. Results were used to identify primary exposure, secondary exposure (from re-activation or re-exposure) and development of test-negative latent carrier (TNLC) status. 4153 test results from 828 cow-lactations were analysed. Primary exposure occurred on two herds, secondary exposure occurred on all herds and development of TNLC status occurred in eight herds. Incidence of secondary exposure reduced over time and may have been related to increasing time since parturition. Regular secondary exposure is required to maintain measurable antibody status.

Aspects of bovine herpesvirus-1 infection in dairy and beef herds in the Republic of Ireland

BackgroundInfection with bovine herpesvirus-1 (BHV-1) causes a wide range of disease manifestations, including respiratory disease and abortion, with world-wide distribution. The primary objective of the present study was to describe aspects of BHV-1 infection and control on Irish farms, including herd-level seroprevalence (based on pooled sera) and vaccine usage.MethodsThe characteristics of a diagnostic indirect BHV-1 antibody ELISA test when used on serum pools were evaluated using laboratory replicates for use in the seroprevalence study. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. A proposed cut off (PCO) PP was applied in a cross-sectional study of a stratified random sample of 1,175 Irish dairy and beef cattle herds in 2009, using serum pools, to estimate herd seroprevalence. The study was observational, based primarily on the analysis of existing samples, and only aggregated results were reported. For these reasons, ethical approval was not required. Bulk milk samples from a subset of 111 dairy herds were analysed using the same ELISA. Information regarding vaccine usage was determined in a telephone survey.ResultsA PCO PP of 7.88% was determined to give 97.1% sensitivity and 100% specificity relative to the use of the ELISA on individual sera giving maximization of the prevalence independent Youden's index, on receiver operating characteristics analysis of replicate results. The herd-level BHV-1 seroprevalence was 74.9% (95% CI - 69.9%-79.8%), with no significant difference between dairy and beef herds. 95.5% agreement in herd classification was found between bulk milk and serum pools. Only 1.8 percent of farmers used BHV-1 marker vaccine, 80% of which was live while 75% of vaccinated herds were dairy.A significant association was found between herd size (quartiles) and seroprevalence (quartiles).ConclusionsThe results from this study indicate BHV-1 infection is endemic, although BHV-1 vaccines are rarely used, in the cattle population in Ireland.

Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits β interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels

Bovine herpesvirus 1 (BHV-1), an alpha-herpesvirinae subfamily member, establishes a life-long latent infection in sensory neurons. Periodically, BHV-1 reactivates from latency, infectious virus is spread, and consequently virus transmission occurs. BHV-1 acute infection causes upper respiratory track infections and conjunctivitis in infected cattle. As a result of transient immune-suppression, BHV-1 infections can also lead to life-threatening secondary bacterial pneumonia that is referred to as bovine respiratory disease. The infected cell protein 0 (bICP0) encoded by BHV-1 reduces human β-interferon (IFN-β) promoter activity, in part, by inducing degradation of interferon response factor 3 (IRF3) and interacting with IRF7. In contrast to humans, cattle contain three IFN-β genes. All three bovine IFN-β proteins have anti-viral activity: but each IFN-β gene has a distinct transcriptional promoter. We have recently cloned and characterized the three bovine IFN-β promoters. Relative to the human IFN-β promoter, each of the three IFN-β promoters contain differences in the four positive regulatory domains that are required for virus-induced activity. In this study, we demonstrate that bICP0 effectively inhibits bovine IFN-β promoter activity following transfection of low passage bovine cells with interferon response factor 3 (IRF3) or IRF7. A bICP0 mutant that localizes to the cytoplasm inhibits bovine IFN-β promoter activity as efficiently as wt bICP0. The cytoplasmic localized bICP0 protein also induced IRF3 degradation with similar efficiency as wt bICP0. In summary, these studies suggested that cytoplasmic localized bICP0 plays a role in inhibiting the IFN-β response during productive infection.

Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1)

The E2F family of cellular transcription factors controls cell cycle progression and cell death. During cell cycle progression, activated cyclin-dependent kinases phosphorylate the retinoblastoma (Rb) protein, causing the release and activation of E2F family members. Previous studies demonstrated that bovine herpes virus 1 (BHV-1) productive infection increases E2F1 protein levels, the bICP0 early promoter is activated more than 100 fold by E2F1 or E2F2, and silencing E2F1 reduced the efficiency of productive infection. In this study, the effect of herpes simplex virus type 1 (HSV-1) productive infection on E2F protein levels and regulation of E2F dependent transcription was compared to BHV-1 infection in the same permissive cell line, rabbit skin (RS) cells. Silencing E2F1 with a specific siRNA reduced HSV-1 productive infection approximately 10 fold in RS cells, and total E2F1 protein levels increased during productive infection. In contrast to RS cells infected with BHV-1, a fraction of total E2F1 protein was localized to the cytoplasm in HSV-1 infected RS cells. Furthermore, E2F1 did not efficiently trans-activate the HSV-1 ICP0 or ICP4 promoter. When RS cells were transfected with an E2F reporter construct or the cyclin D1 promoter and then infected with BHV-1, promoter activity increased after infection. In contrast, HSV-1 infection of RS cells had little effect on E2F dependent transcription and cyclin D1 promoter activity was reduced. In summary, these studies indicated that silencing E2F1 reduced the efficiency of HSV-1 and BHV-1 productive infection. However, only BHV-1 productive infection induced E2F dependent transcription.

Seroprevalence and risk factors associated to Bovine Herpesvirus 1 (BHV-1) infection in non-vaccinated dairy and dual purpose cattle herds in Ecuador

A cross-sectional study was carried out to determine the seroprevalence and risk factors associated to Bovine Herpesvirus 1 (BHV-1) infection in non-vaccinated dairy and dual purpose cattle herds from Ecuador. A total of 2367 serum samples from 346 herds were collected from June 2008 through February 2009. A questionnaire, which included variables related to cattle, health, management measures and environment was filled out in each herd. A commercial indirect ELISA test was used to determine the seropositivity against BHV-1. Generalized Estimating Equations (GEE) model was used to determine risk factors at individual level, including herd as random effect. The individual seroprevalence to BHV-1 in Ecuador was 43.2% (1023/2367; CI 95%: 41.2–45.2%). The herd prevalence was 82.1%; (284/346; CI 95%: 78.1–86.1%) and the intra-herd prevalence ranged from 12.5 to 100% (mean = 64.1%). The GEE model showed that animal age (>4 years) (OR: 1.44; CI 95%: 1.18–1.75), BRSV infection (OR: 1.45; CI 95%: 1.09–1.92), altitude over the sea level (≤1800 m) (OR: 2.97; CI 95%: 2.1–4.22) and average slope (>11%) (OR: 1.45; CI 95%: 1.07–1.95) are risk factors associated with BHV-1 infection, while a good cleaning of the facilities (OR: 0.66; CI 95%: 0.44–0.99) was shown to be a protective factor.

Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes

In contrast to mice or humans, cattle contain three beta interferon (IFN-β) genes with distinct transcriptional promoters suggesting IFN-β gene expression is not stimulated the same by different viruses. To test this hypothesis, we compared expression of the three IFN-β subtypes after infection with a RNA virus, Sendai, versus a large DNA virus, bovine herpesvirus 1 (BHV-1). Infection of low passage bovine kidney (BK) or established bovine kidney cells (CRIB) with Sendai virus has consistently led to high levels of IFN-β1 RNA. Conversely, infection of CRIB cells, but not BK cells, with BHV-1 increased IFN-β3 RNA levels and to a lesser extent the other two IFN-β subtypes. Inhibition of de novo protein synthesis with cycloheximide resulted in higher levels of IFN-β1 and IFN-β2 RNA levels after BHV-1 infection. Further studies demonstrated that BHV-1 immediate early and/or early genes were primarily responsible for inhibiting the IFN response in BK cells. The three bovine IFN-β promoters were cloned upstream of a reporter gene construct, and their properties analyzed in transient transfection assays. Only the IFN-β3 promoter was trans-activated by IRF3 (interferon responsive factor 3). IRF7 and double stranded RNA (polyI:C) stimulated IFN-β1 and IFN-β3 promoter activity, but not IFN-β2. Relative to the human IFN-β promoter, the IFN-β3 promoter contained fewer nucleotide differences in the positive regulatory domain III (PRD III), PRD IV, and PRD I compared to the IFN-β1 and IFN-β2 promoter. Collectively, these studies provide evidence that virus infection differentially stimulates expression of the three bovine IFN-β genes.

Serological Evidences of Bovine Herpesvirus-1 Infection in Bovines of Organized Farms in India

Bovine herpesvirus type-1 (BHV-1) is an important pathogen of cattle causing a variety of clinical signs, including the upper respiratory tract infection, infectious bovine rhinotracheitis (IBR). Infectious bovine rhinotracheitis, a highly infectious disease of cattle and buffaloes, occurs throughout the world including India. The present study based on micro-serum neutralization test reports the sero-epidemiology of BHV-1 infection in cattle and buffaloes from different parts of India. Serum samples from cattle, bulls, buffalo bulls and yaks were screened for BHV-1 antibodies. A total of 1115 serum samples were screened, and a total of 437 (39.2%) serum samples were found positive and 678 (60.8%) serum samples were found negative. Overall 168 (38.0%) cattle, 17 (85.0%) buffalo, 212 (38.6%) bulls, 8 (13.5%) buffalo bulls and 32 (71.1%) yaks were found positive for BHV-1 antibodies. State wise, Assam had highest seropositivity of 71.1% for yaks, Madhya Pradesh had 68.9% for cattle and Meghalaya was negative for the presence of antibodies to BHV-1 in cattle.

The Chemokine IL8 Is Up-Regulated in Bovine Endometrial Stromal Cells by the BoHV-4 IE2 Gene Product, ORF50/Rta: A Step Ahead Toward a Mechanism for BoHV-4 Induced Endometritis1

Postpartum infections of the endometrium and metritis are common causes of delayed conception and infertility in cattle. These infections are characterized by inflammation of the endometrium and secretion of the chemokine interleukin 8 (IL8), which attracts granulocytes to the endometrium. Bovine herpesvirus 4 (BoHV-4) is tropic for the endometrium and the only virus consistently associated with postpartum metritis. The BoHV-4 Immediate Early 2 (IE2) gene is the first viral gene transcribed by host cells after infection, and the IE2 gene product, ORF50/Rta, transactivates host cell genes. The present study tested the hypothesis that ORF50/Rta transactivates the IL8 gene promoter during BoHV-4 infection of bovine endometrial stromal cells (BESCs). Infection of primary BESCs with BoHV-4 stimulated IL8 gene promoter activity and IL8 protein secretion. However, IL8 production was dependent on the transcription of viral genes, because psoralen/ultraviolet cross-linking of the viral DNA abrogated the response to BoHV-4 infection. Furthermore, IL8 promoter serial deletion analysis revealed a specific region responsive to ORF50/Rta. These observations may represent an endometrial defense mechanism against viral infection or a virulence mechanism by which viral replication stimulates chemokine secretion to attract more susceptible host cells to the endometrium.

Seroepidemiology of bovine herpesvirus 1 (BHV1) infection among Estonian dairy herds and risk factors for the spread within herds

The objectives of this study were to reassess the herd level and within-herd prevalence of bovine herpesvirus 1 (BHV1) infection in Estonian dairy cattle, estimate the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for bulk tank milk (BTM) testing and determine the risk factors related to high prevalence of the infection in herds. To estimate the herd prevalence, BTM samples from each of the 1,205 herds that sell milk to dairy companies were analysed for BHV1 antibodies. One hundred and three herds with known BHV1 infection status were selected to estimate within-herd prevalence and to calculate the sensitivity and specificity of BTM ELISA. In these herds serum samples were collected from cows and youngstock, together with BTM samples. A commercial blocking ELISA test was used to analyse samples for antibodies against BHV1. A questionnaire was completed to collect herd data. The sensitivity and specificity of the BTM ELISA were 76.5% and 97.2%, respectively, and the true herd prevalence of BHV1 was calculated to be 22.0%. The herd prevalence increased significantly with herd size, being 3.4% in the smallest category (less than 20 cows) and 85.7% in herds of size over 400. The mean within-herd prevalence was 37.8% (range 1–100, median 31.5). The mean within-herd prevalence increased with herd size. Data from 59 infected herds was used to determine the risk factors associated with high within-herd prevalence (>50%) of BHV1, using logistic regression analysis. As, in some infected herds, the youngstock were uninfected, risk factors for the presence of BHV1 among youngstock from 6 months until calving were analysed. The results indicate the importance of iatrogenic spread of the virus, since the overall within-herd prevalence was higher in those herds in which a veterinarian was an employee of the farm and an inseminator worked only for the particular farm. The presence of bovine viral diarrhoea virus (BVDV) in a herd was associated with a higher prevalence of BHV1.

Erratum to: Serological evidence of bovine herpesvirus-1 (BoHV-1) infection in yaks (Peophagus grunniens) from the National Research Centre on Yak, India

Erratum to: Serological evidence of bovine herpesvirus-1 (BoHV-1) infection in yaks (Peophagus grunniens) from the National Research Centre on Yak, India

Construction of multimerized scFv that neutralize Bovine Herpes Virus-1 (52.12)

Abstract Our laboratory earlier constructed functional antibody fragments, scFv (single chain fragment variable), with an 18 amino acid linker, against bovine herpes virus-1 (BoHV-1) (Koti et al. 2010. Molec. Immunol. in press). With an objective to enhance scFv avidity, the scFv in Vl-linker-VH configuration was constructed with a short flexible amino acid linker. An overlap PCR product comprising Vl-linker-VH was cloned into pPICZα vector and expressed in Pichia pastoris, under the influence of inducible AOX1 promoter. Affinity purified scFvs were analyzed by Western blot and mass spectrometry that confirmed the expected molecular weight of the scFv. Further, the multimerized scFvs, both purified and unpurified, neutralized BoHV-1 infection of MDBK cells in vitro. Taken together with previous studies, the scFvs with varying linker size are capable of effectively neutralizing BoHV-1. The scFv against BoHV-1 will provide protection at respriratory mucosal surfaces and prevent infection. In conclusion, scFvs both in multimerized and non-multimerized form provide a useful diagnostic and therapeutic agent against an important cattle pathogen. (Supported by NSERC Canada)

Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1

Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1 antigens.